The object of the present invention is a method for the radioimmunoassay of small molecules, such as peptide hormones.
In a radioimmunoassay (RIA) the quantity of a component is determined by adding to a sample the radioactive form of the same substance and the antibody of the substance to be studied. Hence, the component subject to investigation and the added radioactive substance react with the antibody, whereby the higher the proportion of the complex that is radioactive, the lower is the concentration of the studied substance in the sample. This results from the fact that so little of the antibody has been added that all of the substance to be studied and of its radioactive form (antigen) cannot react, for which reason the radioactive and the non-radioactive form compete with each other. By measuring the radioactivity of the complex or of the free form or of both, it is possible to calculate the quantity of the component to be studied in the sample.
The most difficult step of work in radioimmunoassays is the separation of the complex and the free antigen from each other with a sufficiently high precision by means of methods suitable for mass working. There are several techniques in use, depending on the antigen and the operator. A brief summary will be given of these methods of separation:
1. Electrophoresis requires a lot of active working contribution and the sample size is limited (maximum limit 200 .mu.l).
2. Gel filtering requires a lot of active work and time.
3. Non-specific precipitation by means of protein precipitants also encloses free antigen inside the precipitate and requires several washing steps.
4. Immunoprecipitation comprises precipitating of the complex by means of an antibody of the antibody. The precipitate produced is difficult to handle carefully enough, i.e. it can be lost with the washing liquids.
5. By means of adsorption onto a solid phase it is possible to remove the unbound hormone out of the sample and to calculate the complex retained in the liquid phase.
6. The antibody in the solid phase is allowed to react with the antigen, and the free component is washed off.
Among the listed methods, the last two listed are becoming increasingly used. They, however, involve certain limitations. For example, in adsorption onto a solid phase the total protein content of the mixture is critical and must be standardized precisely. The methods also require 2 to 3 precise steps of dosage. Moreover, when hormone contents are measured when determining reasons for disturbed states of metabolism, it is often necessary to choose different separation methods for different hormones. There are no standard methods, and for this reason clinical-chemical laboratories must have high and wide-range specialist knowledge.